Cestoda: Rhinebothriidea) from Stingrays of the Genus Fontitrygon from Senegal

Institute of Parasitology, Biology Centre CAS Folia Parasitologica 2018, 65: 014 doi: 10.14411/fp.2018.014 http://folia.paru.cas.cz

Research Article Two new species of Stillabothrium (Cestoda: Rhinebothriidea) from stingrays of the genus Fontitrygon from Senegal

Elsie A. Dedrick1, Florian B. Reyda1, Elise K. Iwanyckyj1, Timothy R. Ruhnke2

1 Biology Department & Biological Field Station, State University of New York, College at Oneonta, Ravine Parkway Oneonta, NY, USA; 2 Department of Biology, West Virginia State University, Institute, WV, USA

Abstract: Morphological and molecular analyses of cestode specimens collected during survey work of batoid elasmobranchs and their parasites in Senegal revealed two new species of the rhinebothriidean cestode genus Stillabothrium Healy et Reyda 2016. Stillabothrium allisonae Dedrick et Reyda sp. n. and Stillabothrium charlotteae Iwanyckyj, Dedrick et Reyda sp. n. are both described from Fontitrygon margaritella (Compagno et Roberts) and Fontitrygon margarita (Günther). Both new cestode species overlap in geographic distribution, host use and proglottid morphology, but are distinguished from each other, and from the other seven described species of Stillabothrium, on the basis of their pattern of bothridial loculi. Phylogenetic analyses based on sequence data for 1,084 bp from the D1–D3 region of 28S rDNA that included multiple specimens of both new species and eight other species of Stillabothrium corroborated the morphologically-determined species boundaries. The phylogenetic analyses indicate that S. allisonae sp. n. and S. charlotteae sp. n. are sister species, a noteworthy pattern given that the two species of the stingray genus Fontitrygon they both parasitise, F. margaritella and F. margarita, are also sister species. Although species of Stillabothrium vary widely in their patterns of facial loculi, the variation does not appear to correlate with phylogeny. Most species of Stillabothrium parasitise myliobatiform elasmobranch genera of the Dasyatidae Jordan. This study brings the number of described species of Stil- labothrium to nine, three of which occur in the eastern Atlantic, two of which occur off the northern coast of Australia, and four of which are from coastal Borneo. Keywords: tapeworms, taxonomy, 28S rDNA, phylogeny, survey, sister species, biodiversity, species boundaries

Our understanding of the species diversity of many single species. Except for Sungaicestus, each of these gen- orders of cestodes has expanded substantially in recent era is potentially more diverse; in both studies (Reyda et al. years owing to a global effort by an international team of 2016, Caira et al. 2017) the authors referred to specimens scientists to survey, inventory and describe new species. of additional undescribed species of the new genera. This progress was recently summarised in a volume ed- This paper focuses on two previously undescribed spe- ited by Caira and Jensen (2017) in which taxonomic and cies of Stillabothrium that were obtained during field work systematic information on each of the 19 cestode orders on the coast of Senegal in 2003, 2004 and 2005, and re- is provided by the experts of the respective orders. As part ferred to in the study by Healy et al. (2009) as Rhinebothri- of this effort, the generic and species diversity the cestode inae New genus 3 sp. n. 1 and Rhinebothriinae New genus order Rhinebothriidea Healy, Caira, Jensen, Webster et 3 sp. n. 2 (or simply New genus 3 sp. n. 1 and New genus 3 Littlewood, 2009 has been substantially expanded since its sp. n. 2). The two new species described in this study over- establishment in 2009 (Healy et al. 2009) to accommodate lap in geographic distribution and in host use. Both species newly discovered taxa with unique scolex morphologies. were found in the spiral intestines of Fontitrygon margar- Four of the rhinebothriidean genera originally referred ita (Günther) and Fontitrygon margaritella (Compagno et to by Healy et al. (2009) as potentially new, based on both Roberts) and there were several instances of concurrent in- morphological and molecular data, were subsequently de- fections. In this study, as in the one by Reyda et al. (2016), scribed in two studies (Reyda et al. 2016, Caira et al. 2017). morphological data were complemented by sequence data These genera are Stillabothrium Healy et Reyda, 2016, for the D1–D3 region of the 28S rDNA gene. Both sources with seven species, Barbeaucestus Caira, Healy, Marques of data played a key role in delineating the boundaries of et Jensen, 2017 with four species, Divaricobothrium Cai- the new species in the context of a phylogeny of species of ra, Healy, Marques et Jensen, 2017 with two species, and Stillabothrium. Sungaicestus Caira, Healy, Marques et Jensen, 2017 with a

Address for correspondence: F.B. Reyda, Biology Department & Biological Field Station, State University of New York, College at Oneon- ta, Ravine Parkway Oneonta, NY, 13820-4015, USA. Phone: +1-607.436.3719; Fax: +1-607.436.3646; E-mail: [email protected] Zoobank number for article: urn:lsid:zoobank.org:pub:7FD2D9BE-83F5-4051-B13A-6893349BD3DF

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MATERIALS AND METHODS Czech Republic; LRP, Lawrence R. Penner Parasitology Collec- The cestodes described here came from two individuals of tion, Department of Ecology & Evolutionary Biology, University Fontitrygon margarita (Collection Code and Numbers SE-232 of Connecticut, Storrs, Connecticut, U.S.A.; MNHN, Muséum and SE-241) and eight individuals of Fontitrygon margaritella National d’Histoire Naturelle, Paris, France; USNM, United (Collection Code and Numbers SE-125, SE-228, SE-229, SE- States National Museum, Smithsonian Institution, Washington, 233, SE-262, SE-279, SE-306 and SE-308). The stingray speci- D.C. U.S.A. Nomenclatural acts in this manuscript are registered mens were collected from the coast of Senegal during field work at Zoobank.org. that occurred in 2003, 2004 and 2005. Stingrays were collected in The molecular analyses included newly sequenced specimens conjunction with local fishermen. Each host was identified in the and sequences from GenBank that were generated for previous field, assigned a Collection Code and unique Collection Number, studies (Healy et al. 2009, Reyda et al. 2016). All newly se- and photographed, and relevant information (e.g. sex, size) was quenced specimens originally fixed in 95% were initially cut and recorded. A tissue sample was also collected for subsequent DNA either the scolex and/or terminal proglottid was removed and pre- analysis. Additional data for each host specimen can be accessed pared as whole mounts as described above, and deposited in the at the Global Cestode Database (Caira et al. 2012) at www. elas- LRP as hologenophores, The remaining portion of each specimen mobranchs.tapewormdb.uconn.edu by entering its assigned was subjected to the molecular protocols mentioned below. Collection Code and Collection Number (e.g. SE-232). Elasmo- Table 1 provides taxon name, host, collection locality, mu- branch classification follows Naylor et al. (2012a); elasmobranch seum accession number for hologenophores (sensu Pleijel et al. taxonomy follows Last et al. (2016). Field identifications of host 2008) and GenBank accession numbers for each of the specimens specimens were verified using NADH2 sequence data (see Nay- included in the molecular analyses. Sequence data were generated lor et al. 2012b). for two or more individuals of both new species of Stillabothrium In the case of each host specimen, the spiral intestine was and included in the molecular analysis, given that the goal was to removed and opened with a longitudinal incision. A subsample test species boundaries of the new species within the context of a of worms was removed, washed in seawater and sorted into two phylogenetic hypothesis of members of the genus Stillabothrium. subsets. The first subset was fixed in 10% seawater-buffered -for Sequence data obtained from GenBank for the analysis consisted malin and subsequently stored in 70% ethanol; the other subset of a single sequence for each of the seven described species of was fixed in 95% ethanol. Spiral intestines were fixed in 10% sea- Stillabothrium as well as for a specimen of an undescribed spe- water-buffered formalin and additional worms were removed un- cies, referred to as Stillabothrium sp. n. 4 by Reyda et al. (2016). der a dissecting microscope upon return to the laboratory. Worms Protocols for DNA extraction, PCR amplification, DNA sequenc- prepared as whole mounts were hydrated in a graded series of eth- ing, sequence analysis and sequence alignment are as given in anols, stained in Delafield’s hematoxylin, destained in 70% acid Reyda et al. (2016). ethanol, neutralised in 70% basic ethanol, dehydrated in a graded Phylogenetic analysis was conducted on sequences of a total ethanol series, cleared in methyl salicylate, and mounted on glass of 19 specimens of 12 cestode species (Table 1). Anthocephalum slides in Canada balsam. Worms examined with SEM (scanning michaeli Ruhnke et Seaman, 2009 and Escherbothrium sp. were electron microscopy) were cut in half and the strobila of each used as outgroup species. Bayesian inference was conducted us- was prepared as a whole mount as described above to serve as a ing MrBayes version 3.2 (Ronquist and Huelsenbeck 2003) with voucher, and the scolex was examined with SEM. Scoleces were the following settings: lset nst = 6 rates = invgamma ngammacat hydrated in a graded ethanol series, placed in 1% osmium tetroxide = 4; ngen = 5,000,000; samplefreq = 1,000. Fifty percent of the overnight, dehydrated in a graded ethanol series, transferred to samples were discarded on burnin. Bootstrap analysis was also hexamethyldisilazane for 15 min in an exhaust hood and allowed conducted using PAUP* verion 5.4.0b (Swofford 2000). One to air dry. Dried worms were mounted on carbon tabs (Ted Pella, thousand replicates (1,000) were performed, with ten step-wise Inc., Redding, California) on aluminium stubs, placed in a dessi- addition heuristic searches per replicate. cator overnight, sputter coated with 250–300 Å of gold/palladium and examined with a LEO/Zeiss DSM982 Gemini or FEI Nova RESULTS Nano 450 (University of Connecticut) field emission scanning electron microscope (FESEM). Microthrix terminology follows Phylogenetic analyses Chervy (2009). The topology of the tree resulting from the Bayesian Measurements of whole mounted cestodes were obtained us- analysis of the sequence data is given in Fig. 1. The ten ing an ocular micrometre on an Olympus CX31 compound mi- species of Stillabothrium that were analysed in this study croscope, or taken with the aid of LAS V3.8 (Leica Application grouped in two principle clades that correspond to the Suite, Leica microsystems, Switzerland) digital microscopy soft- clades termed Clade 1 and Clade 2 by Reyda et al. (2016). ware connected to a Leica DSC295 digital camera on a Leica Clade 1 and Clade 2 each consist of five species of Stil- DM2500 compound microscope. All measurements are report- labothrium. Clade 1 consists of Stillabothrium allisonae ed in micrometres unless otherwise stated, and are presented in sp. n., Stillabothrium charlotteae sp. n., Stillabothrium descriptions as the range followed in parentheses by the mean, jeanfortiae Forti, Aprill et Reyda, 2016, Stillabothrium standard deviation and number of worms measured. Scolex mor- cadenati (Euzet, 1954) Healy et Reyda, 2016 and the un- phological shape terminology follows Clopton (2004). Drawings described species referred to as Stillabothrium sp. n. 4 were made with the aid of a drawing tube. Museum abbreviations (see Fig. 1). Four of these five species conspicuously lack are as follows: IPCAS, Institute of Parasitology of the Biology marginal loculi on the bothridia, a feature that is a charac- Centre of the Czech Academy of Science, České Budějovice, teristic of S. charlotteae sp. n. Clade 2 membership con-

Folia Parasitologica 2018, 65: 014 Page 2 of 12 doi: 10.14411/fp.2018.014 Dedrick et al.: Two new cestode species from stingrays s. Ruhnke et al. 2015 Ruhnke et al. 2015 Healy et al. 2009 Reyda et al. 2016 Reyda et al. 2016 Reyda et al. 2016 Reyda et al. 2016 Reyda et al. 2016 Reyda et al. 2016 Healy et al. 2009 present study present study present study present study present study present study Healy et al. 2009 present study Healy et al. 2009 References KM658204 KM658197 FJ177117 KX826836 KX826839 KX826842 KX826847 KX826853 KX826854 FJ177111 MH714528 MH714529 MH714531 MH714534 MH714532 MH714533 FJ177112 MH714534 FJ177114 GenBank Acc. No. (D1–D3 28S rDNA) LRP 8515 LRP 8519 LRP 3917 LRP 8994 LRP 9000 LRP 8995 LRP 8986 LRP 9003 LRP 8999 LRP 3898 LRP 9906 LRP 9907 LRP 9908 LRP 9909 LRP 9910 LRP 9911 LRP 3899 LRP 9933 LRP 3906 LRP Voucher Acc. No. Voucher SE-289.1 BO-100.2 KA-148.1 SE-125 SE-241.1 SE-241.3 SE-262.1 SE-262.4 SE-279.2 SE-306.1 SE-125 SE-279.1 BJ-423 CRP-50 AU-56 KA-182.5 BO-237.1 CMO3-3.5 NT-26 Specimen number BJ-423 CRP-50 AU-56 KA-182 SE-289 BO-100 BO-237 KA-148 CMO3-3 SE-125 SE-241 SE-241 SE-262 SE-262 SE-279 SE-306 SE-125 SE-279 NT-26 Field code Gulf of California, San Jose del Cabo, Mexico Eastern Pacific Ocean, Costa Rica Sea, off Dundee Beach, Northern Timor Australia Territory, Java Sea (Pacific Ocean), off Selakau, Kalimantan, Indonesian Borneo West Atlantic Ocean, off Kafountine, Eastern Senegal Sulu Sea (Pacific Ocean), off Kampung Sabah, Malaysian Borneo Tetabuan, South China Sea, off Mukah, Sarawak, Malaysian Borneo Java Sea (Pacific Ocean), off Singkawang, Kalimantan, Indonesian Borneo West Australia Weipa, Gulf of Cerpentaria, Atlantic Ocean, off Mbour, Eastern Senegal Atlantic Ocean, off Djifere, Eastern Senegal Atlantic Ocean, off Djifere, Eastern Senegal Atlantic Ocean, off Kafountine, Eastern Senegal Atlantic Ocean, off Kafountine, Eastern Senegal Atlantic Ocean, off Kafountine, Eastern Senegal Atlantic Ocean, off Joal, Senegal Eastern Atlantic Ocean, off Mbour, Eastern Senegal Atlantic Ocean, off Kafountine, Eastern Senegal Islands, Arafura Wessel Sea, Australia Locality - (Bleeker)‡ Last, Manjaji (Compagno et (Compagno (Compagno (Compagno (Compagno (Compagno et (Compagno (Linnaeus) (Günther) (Günther) (Bleeker)‡ Last, White et (Bennett)‡ (Last, Manjaji-Matsu (Garman)

(Last, White et (Last, ‡ sp. 1 et Yearsley Roberts)‡ et Roberts) et Roberts) et Roberts) et Roberts) Roberts)‡ et Roberts) Hypanus longus Urotrygon Glaucostegus typus biasa Telatrygon Naylor)‡ Rhinobatos rhinobatos Maculabatis pastinacoides heterura Brevitrygon Pastinachus solocirostris Himantura australis Naylor Fontitrygon margaritella Fontitrygon margarita Fontitrygon margarita Fontitrygon margaritella Fontitrygon margaritella Fontitrygon margaritella Fontitrygon margaritella Fontitrygon margaritella Fontitrygon margaritella Maculabatis astra moto et Pogonoski)‡ Host species

Herzog, Forti, Aprill (Butler, 1987) (Butler, Delgado, Ruhnke et Iwanyckyj, Iwanyckyj, Dedrick et Dedrick et Dedrick et Dedrick et Dedrick et Dedrick et Dedrick et Willsey et Willsey (Euzet, 1954) sp. sp. n. 4† Table 1. Cestode specimens included in the molecular analyses, with taxon names, hosts, localities, field and specimen codes, museum voucher numbers for hologenophores, and GenBank number hologenophores, numbers for analyses, with taxon names, hosts, localities, field and specimen codes, museum voucher 1. Cestode specimens included in the molecular Table Anthocephalum michaeli Seaman, 2009 Escherbothrium Stillabothrium amuletum Healy et Reyda, 2016 Stillabothrium ashleyae Reyda, 2016 Stillabothrium cadenati Healy et Reyda, 2016 Stillabothrium campbelli Dedrick et Reyda, 2016 Stillabothrium davidcynthiaorum Daigler et Reyda, 2016 Stillabothrium hyphantoseptum et Reyda, 2016 Bergman Stillabothrium jeanfortiae et Reyda, 2016 Stillabothrium allisonae Reyda sp. n. Stillabothrium allisonae Reyda sp. n. Stillabothrium allisonae Reyda sp. n. Stillabothrium allisonae Reyda sp. n. Stillabothrium allisonae Reyda sp. n. Stillabothrium allisonae Reyda sp. n. Stillabothrium allisonae Reyda sp. n. Stillabothrium charlottae Dedrick et Reyda sp. n. Stillabothrium charlottae Dedrick et Reyda sp. n. Stillabothrium †Undescribed rhinebothriine species follow the naming scheme used by Healy et al. (2009) ‡Hosts that were referred to under different names in previous studies Species†

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Fig. 1. Phylogram based on Bayesian analysis of a 1,084 bp region of D1–D3 28S rDNA. Numbers above branches indicate Bayesian posterior probabilities, numbers below indicate bootstrap percentage values based on 1,000 replicates. Scale indicates expected number of substitutions per site. Diagrammatic line drawings of fully opened bothridia of the nine described species are shown representing those respective species. Host species information in Table 1. sists of Stillabothrium ashleyae Willsey et Reyda, 2016, of S. charlotteae sp. n. that analysed were from two dif- Stillabothrium davidcynthiaorum Daigler et Reyda, 2016, ferent individuals of F. margaritella and were identical in Stillabothrium campbelli Delgado, Dedrick et Reyda, sequence for the D1–D3 region of 28S rDNA gene. 2016, Stillabothrium hyphantoseptum Herzog, Bergman et Reyda, 2016, and Stillabothrium amuletum (Butler, 1987) Descriptions Healy et Reyda, 2016. Three of the five species in Clade 2, S. amuletum, S. hyphantoseptum, and S. campbelli, pos- Stillabothrium allisonae Dedrick et Reyda sp. n. Figs. 1–3 sess septa that conspicuously overlap one another, a feature lacking in the other two species, S. ashleyae and S. david- ZooBank number for species: cynthiaorum. urn:lsid:zoobank.org:act:D805E908-579C-4A2F-9095-C7AE24EF3BC6 The monophyly of replicate specimens of both new species of Stillabothrium was found as a result of the Bayesi- Description (based on whole mounts of 17 complete an analysis. This arrangement was strongly supported with mature worms and 3 scoleces prepared for SEM): Worms Bayesian posterior probabilities and with bootstrap analy- (Fig. 2A) euapolytic, acraspedote, 1.37–3.11 mm (2.16 sis. The topology observed within the seven individuals of ± 0.48; n =17) long, greatest width 330–639 (457 ± 94; the S. allisonae clade did not correspond to host use. Six of n = 17) at level of scolex; 6–12 (9.4 ± 2; n = 17) proglottids the seven specimens, including one from Fontitrygon mar- per worm. Cephalic peduncle lacking; darkly staining ger- garita (LRP 9906) and five from Fontitrygon margaritel- minative zone present. la (LRP Nos. 3898, 9908–9911), had identical sequences Scolex (Fig. 2B) consisting of scolex proper bearing whereas the seventh specimen, LRP 9907 from F. marga- four stalked bothridia. Stalks 50–125 (82 ± 21; n = 16) long rita, differed from the others by 1 bp. The two specimens by 40–75 (54 ± 10; n = 16) wide, attached slightly posteri-

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Fig. 2. Line drawings of Stillabothrium allisonae sp. n. from Fontitrygon margaritella (Compagno et Roberts) and Fontitrygon margarita (Günther). A – whole worm (Holotype ex F. margaritella; MNHN HEL758); B – scolex (Paratype ex F. margaritella; USNM 1474746); C – terminal proglottid (Paratype ex F. margarita; USNM 1474747). or to middle of bothridia. Bothridia (Fig. 2B) usually con- n = 17) wide; bothridial margins with thin rim of tissue. tracted, varying in shape from shallowly-deltoid (Fig. 3B) Bothridia (Fig. 2B) each with one anterior loculus, middle to very shallowly-deltoid (Figs. 2A, 3A), facially loculated, row of 4 (n = 17) loculi, and posterior row of 7 (n = 17) loc- 195–375 (277 ± 52; n = 17) long by 220–390 (294 ± 48; uli longer than wide. Anterior-most loculus 28–47 (36 ± 6;

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A B

C D E

F G H I

Fig. 3. Scanning electron micrographs of Stillabothrium allisonae sp. n. from Fontitrygon margaritella (Compagno et Roberts). A, B – scoleces, letters indicate locations of other micrographs; C – bothridial rim; D – distal bothridial surface of loculus in anterior region of bothridium; E – proximal bothridial surface and rim; F – proximal bothridial surface; G – distal portion of stalk; H – proximal portion of stalk; I – strobila. n = 17) long by 35–63 (48 ± 6; n = 17) wide. Longitudinal longer than wide. Strobila widest at terminal proglottid; septa of posterior row not overlapping transverse septa of terminal proglottid 480–970 (675 ± 122; n = 17) long anterior region. by 80–210 (123 ± 40; n = 17) wide; genital pore located

Loculi and septa (Fig. 3D) of distal bothridial surfaces 37– 50% (44 ± 5; n = 15) of proglottid length from pro- bearing capilliform filitriches and coniform spinitriches. glottid posterior margin. Immature proglottids 5–11 (7.9 ± Bothridial rim bearing capilliform filitriches (Fig. 3C). 1.8; n = 17) in number. Mature proglottids 1–2 (1.6 ± 0.5; Proximal bothridial surfaces away from rim (Fig. 3E) bear- n = 17) in number. ing acicular filitriches throughout bothridium. Posterior Testes 18–30 (22 ± 3; n = 16) in number, 1 layer deep, half of proximal bothridial surfaces (Fig. 3F) bearing patch arranged in 2 columns (Fig. 2C); columns extending from of coniform spinitriches near, but not extending to, both- anterior margin of proglottid to level of genital pore, 18–42 ridial rim. Bothridial stalks bearing coniform spinitriches (28 ± 6; n = 14) long by 20–60 (34 ± 10; n = 13) wide. on distal portion (Fig. 3G) and only capilliform filitriches Vas deferens coiled, entering anterior margin of cirrus sac, on proximal portion (Fig. 3H). Strobila (Fig. 3I) bearing extending from area anterior to ovarian isthmus to overlap capilliform filitriches only. several posterior-most testes (Fig. 2C), extensive in termi- Strobila with 2–6 (3.8 ± 1.2; n = 17) proglottids wider nal mature proglottid. Cirrus sac thin-walled, oval, extend- than long followed by 4–8 (5.6 ± 1.2; n = 17) proglottids ing medially to midline or near midline of proglottid; cirrus

Folia Parasitologica 2018, 65: 014 Page 6 of 12 doi: 10.14411/fp.2018.014 Dedrick et al.: Two new cestode species from stingrays sac in terminal proglottid 38–67 (55 ± 9; n = 12) long by posterior area of the bothridia with loculi that are longer 28–54 (42 ± 8; n = 12) wide. Cirrus spinitriches present. than wide) of the bothridia. In addition, the anterior region Vagina (Fig. 2C) thick-walled, sinuous, slightly over- of the bothridia of S. allisonae consists of a horizontal row lapping antero-medial portion of cirrus sac in some spec- of four loculi after the single anterior loculus, whereas in S. imens, extending past midline of proglottid from ootype ashleyae and S. davidcynthiaorum it consists of a horizon- region to anterior margin of cirrus sac then laterally to open tal row of two loculi after the single anterior loculus. The into genital atrium anterior to cirrus sac; vaginal sphincter bothridia of S. allisonae differs from those of S. campbelli absent. Seminal receptacle present. Ovary near posterior in that they possess a greater number of loculi in the poste- end of proglottid, H-shaped in frontal view, tetralobed in rior region (7) than in the anterior region (5), and because cross section; ovarian lobes somewhat asymmetrical; poral none of their horizontal and vertical septa overlap one an- and aporal ovarian lobes in terminal proglottids 130–365 other, whereas they do prominently in S. campbelli, and in (228 ± 72; n = 15) and 152–435 (253 ± 79; n = 15) long, S. hyphantoseptum and S. amuletum. respectively. Maximum width of ovary 57–139 (85 ± 24; Stillabothrium allisonae is further distinguished from n = 15). Ovarian isthmus at or near midpoint of ovary; po- S. campbelli in that its cirrus sac only extends medially to ral lobe of ovary stopping 17–52 (30 ± 13; n = 13) short of or near the midline of the proglottid instead of well past genital pore, stopping 8–45 (22 ± 20; n = 3) short of cir- the midline (see fig. 6C in Reyda et al. 2016), as isthe rus sac, or overlapping its posterior portion. Mehlis’ gland case with S. campbelli, and in that its uterus only extends posterior to ovarian isthmus, 27–45 (34 ± 7; n = 11) long as far posteriorly as the ovarian isthmus, instead of to the by 22–38 (29 ± 5; n = 11) wide. Vitellarium follicular; vi- posterior margin of the proglottid, as it does in S. campbel- telline follicles arranged in 1 dorsal and 1 ventral column li. Stillabothrium allisonae can also be distinguished from on each side of proglottid; columns extending from near S. hyphantoseptum in its possession of more testes than the anterior to posterior margin of proglottid, interrupted by latter species (18–30 vs. 9–16). Stillabothrium allisonae terminal genitalia, and interrupted to varying degrees by further differs from S. hyphantoseptum and S. amuletum, ovary (Fig. 2A,C). Uterus ventral, sacciform, extending and from S. cadenati and S. jeanfortiae in the that the loculi from near isthmus of ovary to near anterior margin of pro- in the anterior region of the bothridium are not oriented in glottid. tandem, whereas they are in each of the four latter species. Informal synonyms: New genus 3 sp. n. 1 of Healy et Stillabothrium allisonae can also be distinguished from al. (2009), Caira et al. (2014), Ruhnke et al. (2015) and S. amuletum and S. cadenati in proglottid morphology. In Marques and Caira (2016); Stillabothrium sp. n. 1 of Rey- S. allisonae the cirrus sac extends medially to or near the da et al. (2016). midline of the proglottid, whereas in S. cadenati and S. am- Type host: Fontitrygon margaritella (Myliobatiformes: uletum it extends well past the midline (fig. 12C in Reyda Dasyatidae). et al. 2016 and fig. 22 in Butler 1987, respectively); the Additional host: Fontitrygon margarita (Myliobati- vagina of S. allisonae is not recurved, whereas it is in S. ca- formes: Dasyatidae). denati and S. amuletum; the genital atrium of S. allisonae T y p e l o c a l i t y : Atlantic Ocean off Mbour (14º24’22”N, lacks the convoluted walls and muscular appearance that 16º58’6”W), Senegal (SE-125). characterises the genital atrium of S. cadenati and S. amu- Additional localities: Atlantic Ocean off Joal letum; the vitellarium of S. allisonae is restricted to varying (14º10’30”N, 16º51’12”W) (SE-228, SE-229, SE-306, SE- degrees by the ovary whereas it is not in S. cadenati and 308), Djifere (13º55’50”N, 16º45’52”W) (SE-232, SE-233, S. amuletum. Stillabothrium allisonae also possesses more SE-241), and Kafountine (12º55’41”N, 16º45’10”W) (SE- testes than S. cadenati (18–30 vs. 7–13). 262, SE-279), Senegal. Site of infection: Spiral intestine. Stillabothrium charlotteae Iwanyckyj, Dedrick et Reyda, T y p e m a t e r i a l : Holotype MNHN No. HEL758; paratypes: sp. n. Figs. 1, 4, 5 MNHN No. HEL 757; IPCAS No. C-796; LRP Nos. 9894– 9903, 9906–9911 (molecular vouchers) and 9904–9905 (SEM ZooBank number: specimens); USNM Nos. 1474744–1474747. urn:lsid:zoobank.org:act:3A504890-8EE0-446D-8489-678C46C6FB64 E t y m o l o g y : This species is named in honour of Allison Has- son, best friend of E.A. Dedrick, for her support and friendship. Description (based on whole mounts of 15 complete mature worms, two incomplete worms and three scoleces Remarks. Stillabothrium allisonae sp. n. can be distin- prepared for SEM): Worms (Fig. 4A) euapolytic, acraspe- guished from each of the seven previously reported species dote, 1.71–3.70 mm (0.26 ± 0.62; n =15) long, greatest of Stillabothrium in the unique configuration of loculi on width 330–526 (412 ± 54; n = 17) at level of scolex; 7–12 its bothridia (Fig. 1). In addition, S. allisonae differs from (8.9 ± 2; n = 14) proglottids per worm. Cephalic peduncle four of its congeners in its proglottid morphology. Stil- lacking; darkly staining germinative zone present. labothrium allisonae has a proglottid morphology that is Scolex (Fig. 4B) consisting of scolex proper bearing similar to that of S. ashleyae and S. davidcynthiaorum but four stalked bothridia. Stalks 45–115 (87 ± 21; n = 17) can be distinguished from each of the latter species in the long by 55–100 (72 ± 10; n = 17) wide, attached slightly morphology of its bothridia because it lacks, rather than posterior to middle of bothridia. Bothridia (Fig. 4B) some- possesses, marginal septa in the posterior region (i.e. the what contracted posteriorly, varying in shape from deeply

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Fig. 4. Line drawings of Stillabothrium charlotteae sp. n. from Fontitrygon margarita (Günther). A – whole worm (holotype; MNHN HEL760); B – scolex (paratype; USNM 1474750); C – terminal proglottid (paratype; LRP 9924). deltoid (Fig. 4A) to finely-deltoid (Fig. 4B) to broadly-del- (4.9 ± 0.3; n = 12) marginal loculi, and posterior row of toid, facially loculated, 215–320 (255 ± 32; n = 17) long 9 (n = 16) loculi longer than wide. Marginal loculi extend by 215–284 (249 ± 20; n = 17) wide; bothridial margins from anterior region of bothridia to anterior portion of pos- with thin rim of tissue. Bothridia (Fig. 4B) each with one terior region of bothridium. Anterior-most loculus 28–40 anterior loculus, middle row of two (n = 14) loculi, 4–5 (33 ± 4; n = 15) long by 35–57 (48 ± 6; n = 16) wide.

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A B

C D E F

Fig. 5. Scanning electron micrographs of Stillabothrium charlotteae sp. n. from Fontitrygon margaritella (Compagno et Roberts). A, B, – scoleces, letters indicate locations of other icrographs; C – distal bothridial surface of loculus in anterior region of bothridium; D – proximal surface near bothridial rim; E – proximal bothridial surface; F – strobila.

Longitudinal septa of posterior region not overlapping sac thin-walled, oval, extending medially past midline of transverse septa of anterior region, thinner than those of proglottid; cirrus sac in terminal proglottid 52–88 (71 ± anterior region. 11; n = 15) long by 42–65 (54 ± 9; n = 15) wide. Cirrus Loculi and septa (Fig. 5C) of distal bothridial surfac- spinitriches present. es bearing capilliform filitriches and coniform spinitrich- Vagina (Fig. 4C) thick-walled, sinuous (Fig. 4C), slight- es. Proximal bothridial rim (Fig. 5D) bearing capilliform ly overlapping antero-medial portion of cirrus sac in some filitriches. Proximal bothridial surfaces away from rim specimens, extending past midline of proglottid from bearing acicular filitriches throughout bothridium and, in ootype region to anterior margin of cirrus sac then laterally posterior half (Fig. 5E), patch of coniform spinitriches. to open into genital atrium anterior to cirrus sac, vaginal Bothridial stalks not observed with SEM. Strobila (Fig. 5F) sphincter absent. Seminal receptacle present. Ovary near bearing capilliform filitriches only. posterior end of proglottid, H-shaped in frontal view, te- Strobila with 1–5 (3.5 ± 1.5; n = 14) proglottids that are tralobed in cross section; ovarian lobes somewhat asym- wider than long followed by 4–7 (5.3 ± 0.9; n = 14) pro- metrical; poral and aporal ovarian lobes in terminal proglottids that are longer than wide. Strobila widest at termi- glottids 125–420 (281 ± 80; n = 17) and 137–438 (319 ± nal proglottid; terminal proglottid 590–1,170 (903 ± 182; n 102; n = 17) long, respectively. Maximum width of ovary = 16) long by 85–150 (115 ± 16; n = 16) wide; genital pore 65–90 (79 ± 9; n = 17). Ovarian isthmus at or near mid- located 39–49% (44 ± 3; n = 16) of proglottid length from point of ovary; poral lobe of ovary stopping 10–90 (52 ± proglottid posterior margin. Immature proglottids 4–10 21; n = 17) short of genital pore, somewhat overlapping (7.1 ± 1.7; n = 17) in number. Mature proglottids 1–2 (1.7 posterior portion of cirrus sac. Mehlis’ gland posterior to ± 0.5; n = 15) in number. ovarian isthmus, 22–65 (42 ± 13; n = 14) long by 22–36 Testes 22–26 (24 ± 2; n = 17) in number, 1 layer deep, (29 ± 5; n = 14) wide. Vitellarium follicular; vitelline fol- arranged in 2 columns (Fig. 4C); columns extending from licles arranged in 1 dorsal and 1 ventral column on each anterior margin of proglottid to level of genital pore, side of proglottid; columns extending from near anterior 18–55 (31 ± 10; n = 17) long by 21–43 (33 ± 7; n = 17) to posterior margin of proglottid, interrupted by terminal wide. Vas deferens coiled, entering anterior margin of cir- genitalia, and interrupted to varying degrees by ovary (Fig. rus sac, extending from area anterior to ovarian isthmus 4A,C). Uterus ventral, sacciform, extending from near to overlap several posterior-most testes (Fig. 4C). Cirrus isthmus of ovary to near anterior margin of proglottid.

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Informal synonyms: New genus 3 sp. n. 2 of Healy et belli (i.e. only extending as far posteriorly as the ovarian al. (2009), Caira et al. (2014), Ruhnke et al. (2015), and isthmus in S. charlotteae vs. extending to the posterior Marques and Caira (2016); Stillabothrium sp. n. 2 of Rey- margin of the proglottid in S. campbelli). The proglottid da et al. (2016). morphology of S. charlotteae differs from that ofS. amule- Type host: Fontitrygon margarita (Dasyatidae: Myliobati- tum and from S. cadenati in several aspects. The vagina of formes). S. charlotteae is not recurved, whereas it is in S. cadenati Additional host: Fontitrygon margaritella (Dasyatidae: and S. amuletum; the genital atrium of S. charlotteae lacks Myliobatiformes). the convoluted walls and muscular appearance that charac- T y p e l o c a l i t y : Atlantic Ocean off Djifere (13º55’50”N, terises those of S. cadenati and S. amuletum; the vitellari- 16º45’52”W), Senegal (SE-241). um of S. charlotteae is restricted to varying degrees by the Additional localities: Atlantic Ocean off Mbour ovary whereas they are not in S. cadenati and S. amuletum. (14º24’22”N, 16º58’6”W) (SE-125), Joal (14º10’30”N, Stillabothrium charlotteae and S. allisonae overlap geo- 16º51’12”W) (SE-228), and Kafountine (12º55’41”N, graphically, use the same two stingray species as definitive 16º45’10”W) (SE-262, SE-279), Senegal. hosts, and possess relatively similar proglottid morpholo- Site of infection: Spiral intestine. gies. The two species can be readily distinguished, howev- T y p e m a t e r i a l : Holotype MNHN No. HEL760; paratypes: er, in several aspects of the bothridial morphology and in MNHN No. HEL 759; IPCAS No. C-797; LRP Nos. 9920– the extent of their cirrus sacs. Stillabothrium charlotteae 9929, 9933 (molecular voucher) and 9930–9932 (SEM speci- differs fromS. allisonae in its possession of a posterior row mens); USNM Nos. 1474748–1474751. of nine, rather than seven, loculi and also in its possession, E t y m o l o g y : This species is named in honour of Charlotte rather than lack, of a series of four marginal loculi in tan- Winter, grandmother of E.K. Iwanyckyj, for her support of her dem on each side of the bothridium. granddaughter’s studies and journey through life. These differences are less apparent in specimens in which the bothridia are contracted; in such specimens, the Remarks. Stillabothrium charlotteae sp. n. can be dis- number of septa that connect to the anterior-most loculus tinguished from each of its eight congeners in the unique can be used to distinguish the two species (three in S. char- configuration of loculi on its bothridia (Fig. 1). It can also lotteae vs. one in S. allisonae; see Fig. 1). The septa in be distinguished from six of its eight congeners in its pro- S. charlotteae vary in width, with the longitudinal septa in glottid morphology. The pattern of loculi on the bothrid- the posterior region of bothridia being thinner than those ia of S. charlotteae differentiate it from S. ashleyae and septa in the anterior region (Fig. 4B), whereas the septa S. davidcynthiaorum in two aspects. First, the bothridia of in S. allisonae are more uniform in width throughout the Stillabothrium charlotteae bear a row of four loculi im- bothridium (Fig. 2B). Finally, the cirrus sac of S. charlot- mediately behind the anterior-most loculus, but those of teae is more extensive than that of S. allisonae in that it S. ashleyae and S. davidcynthiaorum bear a row of only extends medially past the midline of the proglottid in the two loculi. Second, the marginal loculi of S. charlotteae are former species but only extends to or near the midline in positioned differently than those of S. ashleyae and S. da- the latter species. vidcynthiaorum. Whereas the marginal loculi of S. charlotteae span the anterior half of the bothridia, they are lo- DISCUSSION cated in the posterior half of the bothridia of the latter two Reyda et al. (2016) emphasised the extensive variation species. Stillabothrium charlotteae is further distinguished in the arrangement of facial loculi of the bothridia of the from S. davidcynthiaorum in its possession of more testes seven species of Stillabothrium characterised in that study. than the latter species (22–26 vs. 11–21). The scope of variation in the arrangement of facial loculi The presence of marginal loculi of S. charlotteae are is expanded here with the addition of Stillabothrium allis- sufficient to differentiate it from each of the other six spe- onae and Stillabothrium charlotteae, and is depicted in the cies of Stillabothrium (S. campbelli, S. hyphantoseptum, context of the phylogeny in Fig. 1. Somewhat surprisingly, S. cadenati, S. amuletum, S. jeanfortiae and S. allisonae) but in line with the results of Reyda et al. (2016), there which all lack marginal loculi, but there are additional re- are no explicit phylogenetic patterns of variation in the ar- spective differences between it and the other six species. rangement of facial loculi with respect to the phylogeny. Stillabothrium charlotteae can be clearly distinguished For example, those species with overlapping septa (i.e. from S. campbelli, S. hyphantoseptum, S. cadenati, S. am- Stillabothrium amuletum, Stillabothrium hyphantoseptum, uletum and S. jeanfortiae in that the anterior region of the Stillabothrium campbelli, and to some extent, Stillaboth- bothridium in each of the latter five species consists of loc- rium cadenati) do not form a single clade to the exclusion uli oriented in tandem, unlike those of S. charlotteae. of other species, nor do those species with marginal loc- Stillabothrium charlotteae is further distinguished from uli (Stillabothrium ashleyae, Stillabothrium davidcynthi- S. campbelli, S. hyphantoseptum and S. amuletum in that aorum and S. charlotteae) nor those species with loculi none of its septa overlap one another, whereas they do oriented in tandem (S. campbelli, S. hyphantoseptum, S. so prominently in each of the latter three species. Also, cadenati, S. amuletum and Stillabothrium jeanfortiae). In- S. charlotteae possesses more testes than S. campbelli and stead, both Clade 1 and Clade 2 possess species with each S. hyphantoseptum (22–26 vs. 12–19 and 9–16, respective- of those features. Given the anticipated additional species ly) and has a less extensive uterus than that of S. camp- diversity of Stillabothrium (see below), future studies may

Folia Parasitologica 2018, 65: 014 Page 10 of 12 doi: 10.14411/fp.2018.014 Dedrick et al.: Two new cestode species from stingrays reveal phylogenetic patterns of morphological variation F. margaritella and five specimens of S. allisonae from that remain unclear at present. F. margarita that were measured did not discernibly dif- In addition to scolex morphological diversity, species of fer in their morphology. The conspecificity of specimens Stillabothrium vary extensively in their geographic distri- of S. allisonae from F. margaritella and F. margarita was bution. Species of Stillabothrium occur in the Eastern and further supported by the results of the molecular analyses Western Hemispheres, specifically the Indo-Pacific and in that one of the two specimens from F. margarita had an the eastern Atlantic. Some general patterns of geographic identical sequence to five of the six specimens fromF. mar- variation among species of Stillabothrium are evident with garitella (see Results and Fig. 1) for the D1–D3 region of respect to the phylogeny. Curiously, both clades include 28S rDNA gene. It should be noted that Reyda et al. (2016) species of Stillabothrium from northern Australia (S. amu- also found a somewhat relaxed level of host specificity for letum in Clade 2 and S. jeanfortiae in Clade 1). The other two of the seven species of Stillabothrium examined in that biogeographic areas represented by the species in the phy- study. logeny are restricted to either Clade 1 or Clade 2. All three The two host species F. margaritella and F. margari- species of Stillabothrium from the eastern Atlantic (S. ca- ta were found to be sister species in the analysis by Last denati, S. charlotteae and S. allisonae) are within Clade et al. (2016). This scenario in which two sister species of 2, and all four species from coastal Borneo (S. campbelli, cestode parasitise two sister species of elasmobranch hosts S. hyphantoseptum, S. davidcynthiaorum and S. ashleyae) is of particular interest because it raises questions about occur within Clade 1. patterns of speciation and adaptive radiation. In addition, The pattern of host associations evident from the phy- there are relatively few parallel scenarios that have been logeny of species of Stillabothrium is the same as that documented among elamobranch cestodes. One such sce- found by Reyda et al. (2016); a diversity of batoid host nario exists in the rhinebothriidean genus Anthocephalum species, most of which are dasyatids, are represented in Linton, 1890, in which the sister species Anthocephalum both clades. Clade 1 includes species of Stillabothrium healyae Ruhnke, Caira et Cox, 2015 and Anthocephalum from myliobatiform genera from the Dasyatidae Jordan in- odonnellae Ruhnke, Caira et Cox, 2015 parasitise two cluding Fontitrygon Last, Naylor et Manjaji-Matsumoto, closely related species of Neotrygon Castelnau (Ruhnke et Himantura Müller et Henle and Maculabatis Last, Nay- al. 2015). Given the relatively short list of cases of sister lor et Manjaji-Matsumoto. A single species from Clade 1, species of elasmobranch cestodes parasitising sister spe- S. cadenati, parasitises the rhinopristiform Rhinobatos rhi- cies of elasmobranchs, the host-parasite system character- nobatos (Linnaeus). Likewise, in Clade 2, most of the host ised in this study consisting of two species of Fontitrygon associations are with myliobatiform genera of the Dasyat- and two species of Stillabothrium would make an ideal fo- idae including Maculabatis, Brevitrygon Last, Naylor et cus for future studies of coevolution. The improved taxo- Manjaji-Matsumoto, and Telatrygon Last, Naylor et Man- nomic framework for Stillabothrium that we have provided jaji-Matsumoto, but there is a single host association with here make this an appealing host-parasite system for future the rhinopristiform Glaucostegus typus (Bennett). Given studies of host and parasite evolution to explore. that most of the known species of Stillabothrium parasitise species of dasyatids, and given the high diversity of that Acknowledgements. We are grateful to Janine Caira (University of Connecticut, Storrs, Connecticut) and Kirsten Jensen (Univer- elasmobranch family (see Last et al. 2016), we predict sity of Kansas, Lawrence, Kansas) for their efforts to collect the continued discovery of new Stillabothrium species as more specimens that were utilised in this study during multiple field trips dasyatids are examined, especially those species that oc- to Senegal. Carrie Fyler and Kenneth Barber also assisted with that cur in the eastern Atlantic and Indo-Pacific, i.e. the known field work, as did Claire Healy, who generated some of the SEM distribution of the species Stillabothrium reported to date. images used in this study. We thank Andrew Haslach (West Vir- Stillabothrium allisonae and S. charlotteae are sister ginia State University) for providing the DNA sequence data for S. species with a somewhat relaxed level of host specificity allisonae and S. charlotteae. Kaylee Herzog (University of Kansas, in that they parasitise the same two host species, Fontitr- Lawrence, Kansas) provided helpful comments about these spe- ygon margaritella and Fontitrygon margarita. Given that cies. This work was partially supported with funds from National this somewhat relaxed level of host specificity is unusual Science Foundation (NSF) grants, Partnership for Enhancing Ex- relative to other rhinebothriidean cestodes (e.g. Ruhnke et pertise in Taxonomy (PEET) grant (DEB No. 0118882), a Plane- tary Biodiversity Inventory (PBI) collaborative grant (DEB Nos. al. 2015) which, for the most part, exhibit a strict or oiox- 0818696 and 0818823), and a Field Stations and Marine Labora- enous level of host specificity sensu( Euzet and Combes tories (FSML) grant (DBI No. 1034744). Partial funding for this 1980), one focus of this study was comparison of puta- study was also provided by grants from the State University of tively conspecific specimens from their respective hosts. New York College at Oneonta Foundation, Inc., the SUNY Oneon- Morphological examination of the three specimens of ta Alumni Association, and the SUNY Oneonta Research Founda- S. charlotteae from F. margaritella and the 14 specimens tion. This work was performed in part at the Biosciences Electron of S. charlotteae from F. margarita did not reveal any mor- Microscopy Facility of the University of Connecticut (NSF grant phological differences between specimens from each host No. 1126100). Any opinions, findings, and conclusions or recom- species. Likewise, the 12 specimens of S. allisonae from mendations here are those of the authors and do not necessarily reflect the views of the National Science Foundation.

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Received 27 February 2018 Accepted 17 August 2018 Published online 21 September 2018

Cite this article as: Dedrick E.A., Reyda F.B., Iwanyckyj E.K., Ruhnke T.R. 2018: Two new species of Stillabothrium (Cestoda: Rhinebothriidea) from stingrays of the genus Fontitrygon from Senegal. Folia Parasitol. 65: 014.

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